I. Cytokines and the Acute Phase Response
Infection, injury, trauma, and a variety of other immunological disorders provoke a basic immune system defense response known as the "acute phase response." (Van Snick, J., Annu. Rev. Immunol. 8:253-278 (1990); Akira, S. et al., Immunol. Rev. 127:25-50 (1992); Koj, A. In: The Acute Phase Response to Injury and Infection, Elsevier, Amsterdam, vol. 10, p139 (1985)). The acute phase response is part of the general inflammatory host defense mechanism. The acute phase response is characterized by fever, leukocytosis, negative nitrogen balance, increased vascular permeability, alterations in plasma meta and steroid concentrations, and by an increase in the synthesis of hepatic acute phase proteins (such as .alpha..sub.1 -antitrypsin, (.alpha..sub.1 -antichymotrypsin, and other protease inhibitors, serum amyloid A, and C-reactive proteins) (Van Snick, J., Annu. Rev. Immunol. 8:253-278 (1990)).
Various proteins (termed "cytokines") are involved in mediating the acute phase response. These cytokines include tumor necrosis factor ("TNF"), transforming growth factor-.beta. ("TGF-.beta."), Interleukin-1 ("IL-1"), Interleukin-2 ("IL-2"), Interleukin-8 ("IL-8"), Interleukin-10 ("IL-10") and Interleukin-6 ("IL-6") (Bauer, J. et al., Ann. Hematol. 62:203-210 (1991); Kishimoto, T., Blood 74:1-10 (1989); Akira, S. et al., Immunol. Rev. 127:25-50 (1992); Van Snick, J., Annu. Rev. Immunol. 8:253-278 (1990)).
IL-6 plays a central role in inducing the acute phase response. Its release produces multiple effects. IL-6 induces B cells to synthesize immunoglobulins; it induces fever, promotes the synthesis of corticotropin by the pituitary, stimulates hepatocyte production of the acute phase proteins, and acts as a growth promoter of mesangial cells and keratinocytes. IL-6 exerts its control on acute phase proteins at least in part at the transcriptional level (Morrone, G. et al., J. Biol. Chem 263:12554-12558 (1988)). IL-6 also is believed to play a role in inducing the proliferation of hematopoietic cells, and particularly, cytotoxic T cells.
IL-6 is produced by a large number of cell types, including fibroblasts, endothelial cells, keratinocytes, monocytes-macrophages, T-cells, mast cells, and a variety of tumor cell lines (Van Snick, J., Annu. Rev. Immunol. 8:253-278 (1990); Bauer, J. et al., Ann. Hematol. 62:203-210 (1991)). Accessory cells appear to produce the major source of IL-6, however, significant amounts of IL-6 are also produced by lymphocytes (Hirano, T. et al., Eur. J. Immunol. 18:1797-1801 (1988)).
IL-6 is a protein of 21-28 kD which exhibits extensive post-translational modification. cDNA encoding IL-6 has been cloned, and predicts a precursor protein of 212 amino acids including a hydrophobic signal sequence of 28 residues (Hirano, T. et al., Nature 324:73-76 (1986)). Recombinant human IL-6 can be obtained from Genzyme Corp., Boston, Mass.
IL-6 is secreted into the serum. Normal serum levels of IL-6 are less than 5 pg/ml (Nachbaur, D. M. et al., Ann. Hematol. 62:54-58 (1991)). The protein is not generally produced constitutively by normal cells (Akira, S. et al., Immunol. Rev. 127:25-50 (1992)). Indeed, constitutive expression is a characteristic of a number of pathologic conditions (such as psoriasis, rheumatoid arthritis, cardiac myxoma, multiple myeloma, Castleman's disease, and HIV infection. The level of IL-6 is regulated by positive or negative stimuli. For example, liposaccharides induce cells to produce IL-6; the secretion of glucocorticoids represses IL-6 expression (Akira, S. et al., Immunol. Rev. 127:25-50 (1992)). Other positive inducers of IL-6 production include viruses, interleukin-1 (IL-1), interleukin-3 (IL-3), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), .beta.-interferon, and platelet-derived growth factor. IL-6 production is induced during acute inflammatory processes, and is produced by cells that have been injured.
IL-6 induction rapidly follows injury or trauma. Plasma levels of IL-6 can be detected as early as 30 minutes after incision in patient's undergoing elective surgery (Shenkin, A. et al., Lymphok. Res. 8:123-127 (1989)). Maximal levels of IL-6 are found between 90 minutes and 6 hours post surgery (Pullicino, E. A. et al., Lymphok. Res. 9:2-6 (1990); Shenkin, A. et al., Lymphok. Res. 8:123-127 (1989)). In contrast, upon exposure to an infectious agent, elevated plasma levels may persist for days (Bauer, J. et al., Ann. Hematol. 62:203-210 (1991)). Maximal IL-6 plasma concentrations after sterile trauma, such as elective surgery, are about 100 pg/ml, which is orders of magnitude less than the level (up to 500 ng/ml) associated with bacterial infection (Fiedler, W. et al., Leukemia 4:462-465 (1990); Helfgott, D. C. et al., J. Immunol. 142:948-953 (1989)). Elevated serum levels of IL-6 have been observed in transplant rejection, and inflammatory bowel disease (van Oers, M. H. J. et al., Clin. Exper. Immunol. 71:314-319 (1988); Bauer, J. et al., Ann. Hematol. 62:203-210 (1991)).
II. Assays for Interleukin-6
A. Biological Assays
The observation that a pulse of increased IL-6 synthesis can be observed in the normal response to many kinds of traumatic or infectious events has led to the development of assays for detecting and quantifying serum or urine IL-6 levels.
Since IL-6 has a hybridoma growth promotant activity, the capacity of a patient's serum to support hybridoma growth provides a biological assay for IL-6. Other biological assays exploit the capacity of IL-5 to stimulate the growth of B-cells (Hirano, T. et al, Proc. Natl. Acad. Sci. (U.S.A.) 82:5490-5494 (1985); Yoshizaki, K. et al., Blood 74:1360-1367 (1989)). Indeed, certain B-cell lines require IL-6 as an essential growth factor, and have been used to define sensitive bioassays of IL-6 concentration (Akira, S. et al., FASEB J. 4:2860-2867 (1990); Lansdorp, P. M. et al., Curr. Top. Microbiol. Immun. 132:105-113 (1986); Matsuda, T. et al., Eur. J. Immunol. 18:951-956 (1988); Nijsten, M. W. N. et al., Lancet 2:921 (1987); Van Snick, J. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:9679-9684 (1986); Nachbaur, D. M. et al., Ann. Hematol. 62:54-58 (1991); Ershler, W. B. et al., Lymphok. Cytok. Res. 12:225-230 (1993)).
Sensitivity for assays of IL-6 in the range of 20-100 pg/ml have been reported (Brozik, M. et al., J. Rheumatol. 19:63-68 (1992)). The bioassays require significant incubation times (e.g., 4 days) in order to provide a final result (Nachbaur, D. M. et al., Ann. Hematol. 62:54-58 (1991); Brozik, M. et al., J. Rheumatol. 19:63-68 (1992)).
B. Immunoassays
The availability of antibodies that are capable of specifically binding IL-6 has permitted the development of sensitive immunoassays of IL-6 concentration. Such antibodies can be obtained from Genzyme Corp. (Boston, Mass.), or from R&D Systems, Inc. (Minneapolis, Minn.).
Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target molecule. Immunoassays are used extensively in modern diagnostics (Fackrell, J. Clin. Immunoassay 8:213-219 (1985)). A large number of different immunoassay formats have been described (Yolken, R. H., Rev. Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons, NY (1985); Ngo, T. T. et al., In: Enzyme Mediated Immunoassay, Plenum Press, NY (1985)).
Corcoran, K. A. et al. (Clin. Chem. 37:1046 (1991)) discuss an enzyme immunoassay for the quantification of IL-6 in serum. The assay is stated to be capable of detecting 2.6 pg/ml. A double antibody enzyme-linked immunoassay for IL-6 has been used to determine IL-6 concentrations in synovial fluids (Brozik, M. et al., J. Rheumatol. 19:63-68 (1992)). The assay required only a 1 hour incubation period, however, its sensitivity was 80-fold lower than that of bioassays (i.e. the level of detection was 80 pg/ml). As stated, normal serum levels of IL-6 are less than 5 pg/ml (Nachbaur, D. M. et al., Ann. Hematol. 62:54-58 (1991)). Thus, the double antibody enzyme-linked immunoassay could not be used to evaluate IL-6 serum levels (Brozik, M. et al., J. Rheumatol. 19:63-68 (1992)).
Alternate IL-6 immunoassay protocols have been described by Buyalos, R. P. et al. (Fertil. Steril. 57:1230-1234 (1992)), and by Thavasu, P. W. et al. (J. Immunol. Meth. 153:115-124 (1992)). The assay of Buyalos, R. P. et al. was used to measure IL-6 levels in follicular fluids. The assay's detection limit was 50 pg/ml. The assay of Thavasu, P. W. et al. was used to assay IL-6 in blood, and had a detection level of 70 pg/ml. The assay exhibited problems of stability and irreproducibility (Thavasu, P. W. et al., J. Immunol. Meth. 153:115-124 (1992)).
A commercially available immunoassay tests for IL-6 (IL-6 EASIA, Medgenix Diagnostics) has been described (Soderquist, B. et al., Scand. J. Immunol. 24:607-612 (1992)). The test is based upon an oligoclonal capture antigen system in which several monoclonal antibodies directed against distinct epitopes of IL-6 are used. A solid phase monoclonal immunoassay for IL-6 has also been described (Helle, M. et al., J. Immunol. Meth. 138:47-56 (1991)).
Elevated IL-6 plasma levels have been found in patients in sepsis (Hack, C. E. et al., Blood 74:1704-1710 (1989); Waage, A. et al., J. Exper. Med. 169:333-338 (1989)). However, efforts to correlate the kinetics of IL-6 levels with the severity of septicemia have not yielded clear results. One study, involving S. aureus -induced indicated that the diagnostic value of IL-6 analyses depended upon the availability of other information, such as the C-reactive protein levels in a patient (Soderquist, B. et al., Scand. J. Immunol. 24:607-612 (1992)). A second study of the relationship between IL-6 kinetics and septicemia noted high levels of IL-6 were found in patients experiencing septic shock, but not in patients experiencing both septic shock and meningitis (Waage, A. et al., J. Exper. Med. 169:333-338 (1989)). Even in patients experiencing septic shock, a 1,000-10,000 fold range of IL-6 levels was encountered (Waage, A. et al., J. Exper. Med. 169:333-338 (1989)). A third study of the relationship between IL-6 kinetics and septicemia noted that higher levels of IL-6 were seen in those patients who died from sepsis relative to those who survived, however, no correlation was observed between survival time and IL-6 plasma level (Hack, C. E. et al., Blood 74:1704-1710 (1989)). Indeed, IL-6 levels became undetectable in one patient who ultimately succumbed to sepsis (Hack, C. E. et al., Blood 25 74:1704-1710 (1989)).
As indicated, IL-6 levels rise after elective surgery. The extent of the rise can be correlated to the duration or severity of the surgery (Cruikshank, A. M. et al., Clin. Sci. 79:161-165 (1990)). IL-6 levels for all types of surgery evaluated fell to baseline levels within 4-5 days (Cruikshank, A. M. et al., Clin. Sci. 79:161-165 (1990)). Investigations of the relationship between the change in IL-6 levels and post-surgical trauma have also been conducted. No correlation was found between IL-6 levels and hematopoietic recovery of patients who had received peripheral blood stem cell autographs (Kawano, Y. et al., Blood 81:856-860 (1993)).
Thus, despite the significance of IL-6 in acute inflammatory diseases, and the existing methods for assaying IL-6, such assays have been considered to be of limited diagnostic value (Bauer, J. et al, Ann. Hematol. 62:203-210 (1991)). In particular, IL-6 levels are elevated for only a short period of time, and IL-6 synthesis may be associated with a large number of disease states. Thus, the art indicates that both the absence and the presence of IL-6 levels in serum may be unrelated to a particular injury or trauma (Bauer, J. et al., Ann. Hematol. 62:203-210 (1991)).
As will be appreciated, the capacity to diagnose septicemia or other inflammatory process prior to the onset of life threatening clinical manifestations would be highly desirable. Previous efforts to correlate IL-6 levels with such complications have suggested that changes in IL-6 levels are too transient, and too variable to permit a definitive correlation. A method for using IL-6 levels in serum or other biological fluids in order to diagnose the predisposition of an individual to post-trauma complications would, however, be highly desirable. The present invention provides such a method.